Recombinant DNA technology refers generally to the technique of integrating genetic information from a donor source into vectors for subsequent processing, such as through introduction into a host, whereby the transferred genetic information is copied and/or expressed in the new environment. Commonly, the genetic information exists in the form of complementary DNA (cDNA) derived from messenger RNA (mRNA) coding for a desired protein product. The carrier is frequently a plasmid having the capacity to incorporate cDNA for later replication in a host and, in some cases, actually to control expression of the cDNA and thereby direct synthesis of the encoded product in the host.
This technology has progressed extremely rapidly in recent years, and a variety of exogenous proteins have been expressed in a variety of hosts. By way of example, some of the eukaryotic proteins so-produced include: proinsulin (Naber, S. et al., Gene 21: 95-104 [1983]); interferons (Simon, L. et al., Proc. Nat. Acad. Sci. U.S.A., 80: 2059-2062 [1983] and Derynck, R. et al., Nucl. Acids Res. 1: 1819-1837 [1983]); growth hormone (Goeddel, D., et al., Nature 281: 544-548 [1979]) and mast cell growth factor (Yokota et al., Proc. Nat. Acad. Sci. U.S.A., 81: 1070-1074 [1984]. (These publications and other reference materials have been included to provide additional details on the background of the pertinent art and, in particular instances, the practice of invention, and are all incorporated herein by reference.)
For some time, it has been dogma that the mammalian immune response was due primarily to a series of complex cellular interactions, called the "immune network". While it remains clear that much of the response does in fact revolve around the network-like interactions of lymphocytes, macrophages, granulocytes and other cells, immunologists now generally hold the opinion that soluble proteins (e.g., the so-called lymphokines) play a critical role in controlling these cellular interactions.
Lymphokines apparently mediate cellular activities in a variety of ways. They have been shown to have the ability to support the proliferation and growth of various lymphocytes and, indeed, are thought to play a crucial role in the basic differentiation of pluripotential hematopoietic stem cells into the vast number of progenitors of the diverse cellular lineages responsible for the immune response. Cell lineages important in this response include two classes of lymphocytes: B cells, which can produce and secrete immunoglobulins (proteins with the capability of recognizing and binding to foreign matter to effect its removal), and T cells (of various subsets) that induce or suppress B cells and some of the other cells (including other T cells) making up the immune network.
Research to better understand (and thus potentially treat therapeutically) immune disorders, through the study of B cells, T cells and the other cells involved in the immune response, has been hampered by the general inability to maintain these cells in vitro. However, several immunologists recently discovered that such cells could be isolated and cultured by growing them on secretions from other cells, e.g., conditioned media from splenic lymphocytes stimulated with Concanavalin A (ConA). It has now become clear from this work that the generation of cell clones is dependent on specific factors, such as lymphokines.
Some of the better characterized lymphokines are the so-called interleukins, e.g., lymphocyte activating factor (LAF), which is released from macrophages and can induce replication of thymocytes and peripheral T cells (Mizel, S. et al., J. Immunol. 120: 1497-1503 [1978]), T cell growth factor (TCGF), which was initially detected in conditioned media from lectin-stimulated lymphocytes (Morgan, D. et al., Science 193: 1007-1008 [1976]), and mast cell growth factor (MCGF), which was found in lectin-stimulated T cell clones (Nabel, G. et al., Nature 291: 332-334 [1981]). In 1979, the Second International Lymphokine Workshop labelled LAF as interleukin-1 (IL-1) and TCGF, as interleukin-2 (IL-2). Similarly, although not officially, MCGF is now known generally as interleukin-3 (IL-3) (Ihle, J. et al., J. Immunol. 131: 282-287 [1983]).
In view of the central role T cells play in the immune response, their growth factor, IL-2, has been the subject of considerable study since its discovery about 10 years ago (Smith, K. Immunol. Rev. 51: 337-357 [1980]). IL-2's prime function is almost certainly the stimulation and maintenance of proliferation of most T cell subsets. In fact, the removal of IL-2 from proliferating T cells results in their death within a few hours (Ruscette, F. et al., J. Immunol. 123: 2928-2931 [1977]). IL-2 has also been shown to be one of the lymphokines responsible for cytotoxic T lymphocyte generation, as well as differentiation and induction of function.
Importantly, IL-2's function is not restricted to being just a growth factor for activated T cells. The secretion by T cells of .gamma.-interferon and B cell growth factors appears to be induced by IL-2 (Torres, B. et al., J. Immunol, 126: 1120-1134 [1982] and Howard, M. et al., J. Exp. Med. 158: 2024-2039 [1983]). Indeed IL-2 may be at the center of the lymphokine mediated immune response (for a detailed description of IL-2 activities, see Farrar, J. et al., Immunol. Rev. 63: 129-166 [1982].
Several procedures have been developed for the isolation and purification of IL-2 (e.g., Watson, J. et al., J. Exp. Med. 150: 849-855 [1979]; Engleman, E. et al., J. Immunol. 127: 2124-2127 [1981]; and U.S. Pat. Nos. 4,404,280 and 4,407,945). These procedures generally entail culturing normal spleen cells or certain lymphoma cell lines, and then stimulating the cells with a mitogen. This often results in relatively low concentrations of IL-2 in the cell media, which makes subsequent concentration and purification a formidable task.
Furthermore, the IL-2 preparations almost undoubtedly contain some residual mitogen, as well as contaminating proteins from the IL-2 producing cell line. The development of murine T cell hybridomas producing IL-2 has mitigated the mitogen contamination, but the problem remains that most, if not all, murine IL-2 preparations contain other immunological proteins. These proteins can influence assay results, and thus interfere with the unequivocable determination of the precise range of IL-2 activities.
In the human system, these problems have been basically alleviated by the successful cloning and expression of cDNA's encoding for human IL-2 (Taniguchi, T. et al., Nature 302: 305-310 [1983] and Devos, R. et al., Nucl. Acids Res. 11: 4307-4323 [1983]), but the problems remain in the murine system. Given that most immunological experiments are still performed on mice or mouse-derived cells, research is still greatly hampered.
Indeed, the molecular properties of IL-2 remain uncertain. Although it presently appears that the molecular weight of IL-2 is approximately 30-35,000 daltons (Shaw, J. et al., J. Immunol. 120: 1967-1973 [1978]), at least one investigator believes that murine IL-2 is a dimer made up of two 16,000 dalton components (Caplan, B. et al., J. Immunol, 126: 1351-1354 [1981]). Translation in Xenopus laevis oocytes of size fractionated mRNA (indicating that one or more murine mRNA species of about 1000 to 1100 nucleotides encode a protein exhibiting IL-2 activity) has shed some light on the question (Bleackley, R. et al., J. Immunol. 127: 2432-2435 [1981]), but research has still been slowed by the absence of coding sequences and means for producing large quantities of the desired protein.
Clarification of many of the outstanding issues relating to the molecular biology of murine interleukin-2 requires additional structural data, e.g., substantially full-length sequence analysis of the protein and nucleic acid molecules in question. Protein sequencing offers, of course, a possible means to resolve the matter to a certain degree, but it is very difficult work experimentally and often can provide neither completely accurate nor full-length amino acid sequences. In fact, murine IL-2 appears to be blocked near the NH.sub.2 terminus, rendering protein sequencing even more difficult.
Moreover, having the capability of making bulk quantities of a polypeptide exhibiting murine IL-2 activity (and substantially free from other murine proteins) will greatly facilitate the study of the biology of T cells and other cells involved in the immune response; e.g., by minimizing the necessity of relying on lectin-conditioned media for stimulating cell growth. Accurate and complete sequence data on murine IL-2 will help elucidate the structure of that compound and also serve to simplify the search for other immunological factors. Finally, additional information on any lymphokine will help in evaluating the roles of the various growth factors and cells of the immune network and thus provide insight into the entire immune system -with the concomitant therapeutic benefits.
Thus, there exists a significant need for extensive nucleotide sequence data on the DNAs coding for, and amino acid sequences of, proteins exhibiting murine IL-2 activity, as well as a simple and economic method of making substantial and essentially pure quantities of such materials. The present invention fulfills these needs.